Animal research

Animal models of FAS have allowed researchers to study the mechanisms behind alcohol’s deleterious effects on fetal development. Such models have helped verify hypotheses based on studies of children with FAS and uncover new features of FAS not evident in humans.

.


S

ince the "formal" identification of fetal alcohol syndrome (FAS) in 1973, interest in developing and studying animal models of FAS has swelled, giving rise to a thriving area of research.The development of animal models has provided an invaluable re search tool for characterizing and advanc ing the study of alcohol's adverse effects on the developing fetus.By employing several species (including nonhuman primates) and a variety of methodologies and experimental approaches, animal research has played a major role in advanc ing knowledge of the many immediate and longterm deleterious consequences that follow prenatal alcohol exposur .

Animal research, performed under controlled laboratory conditions, substan tiates hypotheses formed by observing humans with FAS; in other cases, it helps form new hypotheses.Using animal models, researchers have discovered detailed brain abnormalities related to FAS, identified critical periods and criti cal doses of alcohol exposure, and iden tified alcoholinduced biochemical changes in the pregnant animal or her fetuses.These issues are beyond the bounds of clinical FAS research with humans either for ethical reasons or because researchers are unable to exer cise the type of control over the genetic background and environment that is needed for a truly accurate experiment.
After discussing in more detail the benefits of FAS animal models and some related general findings, this article will highlight some of the specific observa tions made by basic animal research in the last 20 years.

Benefits of Animal Models
Animal studies allow researchers a degree of control impossible with research on humans.Researchers can control the genetic background of the parent animals as well as certain environmental factors.Rigorous control may be exerted over variables such as amount of alcohol (i.e., alcohol "dose"), pattern of alcohol con sumption, duration and timing of alcohol exposure during pregnancy, and maternal nutritional status.For example, to identify a threshold dose of alcohol above which fetal damage occurs, alcohol can be ad ministered on particular days or weeks of pregnancy, or the amount of alcohol given can be varied from animal to animal.Fur ther, using animals with different genetic backgrounds may provide insight into the possible contribution of genetic factors that may govern susceptibility to alcohol related damage.

On of the first issues tackled by ani mal researchers was whether alcohol, itself, was responsible for the constellation of birth defects labeled FAS.Alcoholic women frequently abuse tobacco, have poor health, and are malnourished.Any of these factors could result in birth defects, and skeptics questioned the focus on alcohol ingestion alone in the coining of the term "fetal alcohol syndrome."Thus, animal models were used to determine if alcohol alone was a teratogen-a sub stance capable of causing birth defects.

Almos simultaneously, two different laboratories reported that alcohol admin istration to pregnant mice resulted in smaller offspring and birth defects similar to those reported in humans with FAS (Chernoff 1977;Randall et al. 1977).These studies controlled for polydrug use, maternal health, and nutritional factors, indicating that alcohol, per se, is terato genic to humans and mice.

Since he publication of the studies in mice, the teratogenic effects of alcohol have been demonstrated in many other species, including nonhuman primates (National Institute on Alcohol Abuse and Alcoholism [NIAAA] 1990[NIAAA] , 1993)).However, the majority of the research generated with animals has used either mice or rats.Mice have been used primar ily to examine alcoholinduced birth defects (structural malformations), where as rats have been used primarily for studying alcoholinduced behavioral deficits.Indeed, the structural and func tional defects observed in these rodent models of FAS are remarkably similar to those identified in human clinical studies (Driscoll et al. 1990).

Determi ing alcohol's threshold dose and mechanism of action is more compli cated than demonstrating that alcohol is a teratogen.It is highly unlikely that re searchers will identify a single threshold dose or safe limit of prenatal alcohol exposure; a unique, circumscribed, and To make the task even more compli cated, it is likely that different aspects of development may be sensitive to different levels of a cohol.Despite the complexity of the issues, scientists have made inroads into understanding this formidable disor der.Since the identification of FAS two decades ago, animal research has pro duced a wealth of general information regarding the effects of prenatal alcohol exposure on the fetus.Some of these findings are summarized below.


General Findings

Critical Periods.By varying when during pregnancy females are given alcohol, animal research has demonstrated that th

specific type of
irth defect produced depends on the system(s) in the fetus undergoing development at the time of alcohol exposure (Becker 1992;Randall 1987;Webster 1989).Organ systems are most vulnerable to damage by alcohol during the period of most dynamic devel opment.For example, heavy alcohol exposure during the period of craniofacial development (which roughly corresponds to the third and fourth weeks of human pregnancy) will affect the structure of the facial features but not the structure of the kidney, an organ system that develops around the sixth week of human pregnan cy (Webster 1989).This type of exposure and damage can be considered a "binge" type model.

As would be expected, heavy alcohol consumption throughout pregnancy, as opposed to occasional binges, risks the entire constellation of str ctural defects involving several major organ systems and including growth retardation and brain abnormalities (NIAAA 1990(NIAAA , 1993)).This pattern is more consistent with what is known as FAS.

The complexity of alcoholrelated damage is perhaps best illustrated when considering the brain and its sensitivity to alcohol during develo ment.Unlike other organs, the brain is one of the first organ systems to begin to develop and the last to be completed.Thus, the brain appears to be sensitive to the adverse effects of alcohol throughout its development and over all trimesters of pregnancy.Animal research has revealed that some brain regions are more sensitive to the terato genic actions of alcohol and, further, that some cell populations are more vulnerable to alcohol insult than others even within a particular brain region (West 1986).

Thus, critical periods of vulnerability to the harmful effects of alcohol on brain development appear to vary among vari ous regions of the br in.This pattern of damage may explain the wide array and complex pattern of behavioral and neuro logical abnormalities that have been ob served in human and laboratory animal offspring exposed to alcohol at different times during fetal development.


Critical Doses. Animal models have

shown that peak blood alcohol level rather than total amount of alcohol con sumed may represent the "criti al dose," or threshold, of alcohol above which an adverse effect will be seen (West et al. 1990).This finding implies that drinking pattern is critical.Rapid consumption of alcohol in a short period of time will result in a higher blood alcohol level than sipping the same amount of alcohol slow ly over a long period of time.These re sults underscore the importance, when studying humans, of researchers asking women about their pattern of drinking in addition to the total number of drinks, as the former may have a greater negative impact on pregnancy outcome.


Comparing Symptoms of FAS in Animal Models and Humans

The symptoms found in animal models are remarkably similar to those observed in humans (

iscoll et al. 1990).This similarity applies to physica
defects (as illustrated in figure 1) and behavioral effects (table 1).In addition, the deleteri ous effects of in utero alcohol exposure in animal models, as in humans, have been shown to exist along a continuum.Both the fullblown syndrome (FAS) and the partial expression of the syndrome, which has been termed "fetal alcohol effects" (FAE), have been seen in alcoholexposed animals.With adequate animal models, researchers can begin to identify problems not yet identified in humans and the mech anisms of alcohol's effect.


Birth Weight and Growth Patterns

Retarded growth in utero and after birth is a hallmark of FAS and has been reported in a wide variety of species

enatally exposed to alcohol (NIAA
1990(NIAAA , 1993)).In many cases, body weight and body length deficits and small heads (micro cephaly) were dose related and observed after various exposure patterns, although exposure to alcohol during the equivalent of the second and third trimesters ap peared to be most critical for this preg nancy outcome (Becker 1992;Randall 1987;West 1986).In some instances, there was evidence of "catchup" in growth as the animals matured; in others, the growth retardation extended into adult hood (Becker 1992).Some recent reports in mice have indicated that the deficits in growth may not be apparent until adoles cence and early adulthood (Becker 1992).

Animal models allow the use of crossfostering techniques to minimize the possibility that deficient growth pat terns are caused merely by postnatal factors, such as poor maternal behavior, rather than in utero alcohol exposure, per se.With crossfostering, alcoholexposed offspring are raised by control mothers, and control offspring are raised by alcohol exposed mothers.Data from animal stud ies indicate that both reduced birth weight and postnatal growth retardation can be attributed primarily to prenatal alco hol exposure.


Major Organ Systems

As in humans, animal models have demonstrated a wide variety of organ anomalies after acute and chronic expo sure to alcohol (Be

er 1992;Randall 1987
.The organs are most sensitive to damage by alcohol exposure during organogenesis, a period when they are first developing.In rodents, the period of organogenesis includes the second week of a 20 to 21day gestation period (Beck er 1992;Webster 1989).This roughly corresponds to weeks 3 to 8 in human pregnancy.

As mentioned earlier, the time of alcohol exposure within this period deter mines which developing organ system(s) will be affected.Animal studies have m deled these possibilities through care ful control of the timing of alcohol expo sure during embryonic/fetal development (Becker 1992;Webster 1989).

Craniofacial Defects.The presence of characteristic defects of the face and head is important for the diagnosis of FAS in humans.Animals exposed to alcoho

during early organogen
sis are born with a pattern of craniofacial defects that are remarkably similar to the facial features of children with FAS (figure 1) (Sulik et al. 1981;NIAAA 1990NIAAA , 1993)).Further examination of these animal fetuses re vealed other brain malformations, sug gesting that the craniofacial defects may indicate brain abnormalities (Kotch and Sulik 1992).Indeed, a positive relation ship has been noted between the severity of craniofacial defects and mental disabil ity in children with FAS (Streissguth et al. 1991;NIAAA 1990NIAAA , 1993)).

Skeletal Anomalies.Exposure to alcohol during organogenesis results in skeletal malformations in laboratory mice, espe cially limb defects (Becker 1992).Lim malformations primarily have involved the forelimbs, with incomplete growth of one or both forelimbs and various types of defects of the digits on the limbs.The digit defects have been attributed to cell death of the limb buds following alcohol exposure.Other skeletal anomalies report ed include those of the vertebrae, ster num, and ribs (Becker 1992).The pattern of skeletal defects observed in mice is similar to that observed clinically in humans.

Cardiovascular Anomalies.Animals exposed to alcohol at a time that roughly corresponds to as early as weeks 3 to 4 of human pregnancy have had cardiovascu lar ma formations that are similar to those reported in children with FAS (Becker 1992;Webster 1989).Most notably, they include malformations of the heart itself and of the vessels leading to and from the heart.Urogenital Anomalies.In both experimen tal animals and humans, the developing kidney has been shown to be sensitive to alcoholinduced damage (NIAAA 1990(NIAAA , 1993)).Kidney defects were observed in children with FAS only after they were first identified in animals.Renal malforma tions typically have included structural defects of the kidney and the ureter, which carries urine from the kidney to the bladder.Abnormal sexual development and function also have been documented in animals prenatally exposed to alcohol.Many of these effects may be attributed to alcohol induced alteration of sex hormones both at the level of the brain and in the sex organs (discussed below).


Sensory and Motor Effects

A variety of sensory and motor deficits have been identified in children with FAS and modeled in studies with experimental animals.These include visual, auditory, balance, and motor coordination problems.

Visual Deficits.Defects involving the eyes and the visual proce s are common in children with FAS and typically result in impair

visual acuity and nearsig
ted ness.The most common defects are small eyes, drooping eyelids (ptosis), short eye openings, crossed eyes, a reduction in the number of optic nerve axons, and abnor mal vasculature in the retina.A simila pattern of eye anomalies has been found in animal models (NIAAA 1990(NIAAA , 1993)).Investigations of the underlying cause of these defects have shown that embryonic alcohol exposure results in an increased rate of cell death and impaired cell repli cation in the retina as well as reduced myelination (insulation) of the nerve that connects the eye to the brain.The develop ing eye appears to be sensitive to alcohol induced damage.

Auditory Deficits.Children with FAS show a high prevalence of hearing impair ment related to structural damage of the ear during its development.Research with animals has demonstrated that prenatal alcohol exposure can have deleterious effects at various levels of the auditory system.For example, rats prenatally exposed to alcohol have been found to have hearing l ss resulting from both abnormal electrical activity in the brain stem and disruptions in auditory process ing at the level of the cortex (NIAAA 1990(NIAAA , 1993)).

Balance and Motor Deficits.Children with FAS have a wide range of balance and motor coordination deficiencies, some of which persist well into childhood (documented through 12 years of age).They include problems with balance and gait, muscle tremors, and deficits in both gross and fine motor function.Rats pre natally exposed to alcohol have shown delayed developmen

of motor reflexes, deficits
n performance of tasks that re quire motor coordination, and alterations in walking pattern (NIAAA 1990(NIAAA , 1993)).Many of these motor problems suggest damage to the cerebellum, a part of the brain that plays an important role in con trolling motor coordination.Rat studies have confirmed this speculation, as have autopsies of humans with FAS (West 1986).


EFFECTS ON BEHAVIOR


Neonatal and Regulatory Effects

Fetal Movement.The deleterious effects of alcohol exposure have been well char acterized in offspring after birth, but relatively little is known about the behav ior of the alcoholexposed fetus in utero.

Recent procedures involving direct obser vation of rat fetal activity in utero showed a substantial reduction i

ovements compared with non alcoh
lexposed fetuses (NIAAA 1990(NIAAA , 1993)).Such alcoholinduced suppression of prenatal activity also has been shown regarding breathing movements and brain activity in nearterm fetal sheep and in the fetu es of pregnant women who con sumed alcohol (NIAAA 1990(NIAAA , 1993)).Altered fetal activity and reduced fetal movement have been associated with profound effects on morphological devel opment in humans and animals.There fore, alcoholinduced suppression of movement in the developing fetus may be a contributing factor in FAS.

Feeding Behavior.Human newborns prenatally exposed to alcohol are not good feeders.They are easily distracted and fatigued when suckling, and, even when they do feed, they have a weak, irregular suckling response.Similar feed ing abnormalities have been found and systematically studied in rodents pre natally exposed to alcohol (Driscoll et al. 1990).Rat studies suggest hat newborn rats that do not nurse successfully may have impaired ability to detect smell cues, thereby hindering the pups' ability to locate the mother's nipples; difficulty in coordinating motor behaviors; or a damp ened level of arousal to stimuli related to attaching to the nipple.These findings suggest that altered feeding behavior may be related to the growth deficits observed postnatally.If so, basic research efforts can identify ways to improve feeding behavior that may have clinical application.


LongTerm Behavioral Effects

FAS was diagnosed initially in young children, and although abnormal behav iors were observed at birth and during the neonatal period, it was not possible to predict if behavioral problems would get better as the children got older, if they would stay the same, if different types of abnormal behaviors would be observed with maturation, or if the

ildren could be taught to ut
lize strategies that com pensate for their behavior.Given that animals mature significantly faster than humans do, the longterm effects of pre natal exposure to alcohol could be more easily examined in animal models.Now that study populations of humans with FAS are growing older, researchers are finding that abnormal behaviors do persist.The animal models can be used to evaluate the effectiveness of interven tions, particularly drug therapies, that try to alleviate abnormal behaviors.

In animals, many of the behavioral effects of prenatal alcohol exposure have been shown to diminish with age (NIAAA 1990(NIAAA , 1993)).It has been proposed that rather than irreparably altering behavior, alcohol merely retards brain development, with eventual catchup or compensatory behaviors producing more normal devel opment.Recent data, however, have shown the reemergence of some deficits in older animals, suggesting either that the compensatory systems break down with age or that more complex or difficult tasks unmask persistent defects (West et al. 1990;NIAAA 1990NIAAA , 1993)).In particu lar, these deficits include impaired perfor mance in learning and memory tasks.

These findings complement those of clinical studies that have revealed persistent attentional, cognitive, and neurobehavioral deficits in children of alcoholabusing mothers, primarily when the testing con ditions were challenging to the children (Streissguth et al. 1991).

Animal models should prove useful in identifying the types of behavioral deficits that are permanent and t ose that are transient developmental delays.Even more important, however, is the use of animal models to develop effective inter ventions to improve cognitive learning and memory, whether the interventions be pharmacological or behavioral in nature.Such information is crit cal in identifying special education approaches for children exposed to alcohol prenatally so that they can more fully benefit from school.


Effects on the Central Nervous System

Behavioral problems observed in off spring exposed prenatally to alcohol, such as hyperactivity and perseveration (repeti tion of a mental activity with an inability to switch to another activity), poor bal ance and coordination, difficulty walking, and the inability to concentrate or to learn from past experiences, are all indicative of ab

rmal development of the brain, or cent
al nervous system (CNS).Those CNS defects include abnormal structure and connections of brain cells and irregu lar communication from cell to cell.


Animal models should prove useful in identifying the types of behavioral deficits that are permanent and those that are transient developmental delays.

Animal models have demonstrated relationships between abnormal behaviors and specific structural defects in the brain.For example, learning and memory defi cits have been demonstrated in animals that exhibited, on post mortem exa ina tion, alcoholinduced damage in the hip pocampus, a part of the brain that plays an important role in mediating these mental activities (NIAAA 1990(N AAA , 1993)).Also, motor coordination problems have been linked to structural defects in the cerebellum.

Such structurefunction associations observed in animal models may serve to guide similar studies in human FAS/ FAE populations, where more sophisti cated neuropsychological tests may be combined with powerful brainimaging techniques, such as nuclear magnetic reso nance, magnetic resonance imaging, and positron emissions tomography scanning (see the article by .

The discovery of ltered brain chemistry in prenatally alcoholexposed offspring also may help to explain altered behavior.Chemicals in the brain-neurochemicalscontrol brain function and behavior.

Neurochemical systems are involved in the expression of different types of behaviors, including feeding behavior, sleep, and even mental health problems such as depression and anxiety.Animal studies have found that several neuro chemical systems are altered in animals exposed prenatally to alcohol (NIAAA 1990(NIAAA , 1993)).The identification of specific neu ochemical imbalances in animal models and of drugs that alleviate these imbalances may lead to appropriate phar macological interventions for humans with similar behavioral problems.


Effects on Neuroendocrine Systems

Levels of the sex hormones testosterone and estradiol are controlled by a complex feedback system that is critical to main taining the proper balance of these hor mones.The hypothalamus (the part of the brain that converts incoming electrical information from neurons into chemical, bloodborne messengers called hormones) se

s hormonal messages to the pituita
y (the body's master endocrine gland).The pituitary then releases other hormones that act on specific parts of the body, such as the testes (in men) or the ovaries (in women).These target tissues release yet more hormones-testosterone and estradiol, for example-that on reaching standard levels in the blood, trigger the hypothalamus and pituitary gland to stop releasing hormonal messengers.

Disruption of any part of this loop (hypothalamus ➝ pituitary ➝ target tissues ➝ hypothalamus/pituitary) can have detrimental effects.Rat models have shown that prenatal alcohol exposure alters the feedback system in both males and females by altering activity in the critical brain structures and the sex organs (NIAAA 1990(NIAAA , 1993)).As a result, sexual mat ration is delayed, sexual behavior is disrupted, hormone levels are abnormal, and reproductive function is altered.

Abnormal hormonal levels during the critical period of sexual differentiation of the brain may be expected to produce long term effects.In the rat, this critical period corresponds to the end of gestation and extends into the first week of postnatal life (this is equivalent to the third trimester of pregnancy in humans).During this time, the production and secr tion of testos terone are the primary hormonal influ ences that promote the masculinization of the brain in male rat offspring.Prenatal alcohol exposure blunts the normal testos terone surge in male neonates (NIAAA 1990(NIAAA , 1993)), resulting in demasculiniza tion, or feminization, of certain brain structures and behaviors in adult male offspring. 1 This finding might have implications for sexual maturation in humans with FAS (Streissguth et al. 1991).It will be interest ing to see if abnormal sex hormone regula tion and reproductive function observed in animal models predict similar problems in humans exposed to alcohol prenatally.Given that FAS was not identified until 20 years ago, such observations should only now be seen as the newborns who were given a diagnosis of FAS in the early 1970's reach sexual maturation and enter their reproductive years.

Another neuroendocrine anomaly observed in animal models is the biochem ical response to stress in rats exposed to alcohol in utero.The reaction to stress is controlled by a feedback loop similar to that of the sex hormones, involving the hypothalamus, pituitary gland, and the adrenal glands as the target tissue (as opposed to the testes or ovaries).Research findi gs have indicated a heightened response to stress in offspring exposed to alcohol prenatally (NIAAA 1990(NIAAA , 1993)).

Animal models are being used to identify the exact location of the defect in the feedback loop.An aberrant response to stress as a function of exposure to alcohol prenatally has direct clinical implications, especially because alcohol is sometimes consumed to relieve stress.Additionally, maladaptive responses to stress would be expected to interfere negatively with da ly functioning.


EFFECTS ON THE IMMUNE SYSTEM

Animals exposed prenatally to alcohol have demonstrated alcoholinduced defi ciencies in the immune response that might explain the increased risk for infec tions observed in children with FAS.In particular, the animals showed a reduction in the number of cells that fight infection, the Tlymphocytes, which in turn increas 1 The effects obse

ed in females are more variab
e and less clear.

es susceptibility to infection and, possi bly, cancer.Although this area of research has not been studied as extensively as some of the others discussed above, it promises to provide important clinical information related to identification and intervention so that the severity and num ber of infections can be reduced.


STUDIES ON MECHANISMS OF FETAL ALCOHOL DAMAGE

Identifying the mechanism(s) of action by which alcohol affects fetal growth and development is one of the most important roles that animal research can play.Given the multitude of different types of de fects, the involvement of all major organ systems, and

he involvement of many dysfunctional neurochem
cal and bio chemical processes, the search for the underlying mechanism(s) will not be easy.It may be that several different mechanisms are responsible for the myri ad of deleterious effects that follow pre natal alcohol exposure.

Alcohol could affect fetal development in several ways: directly, by damaging and killing the developing fetal cells; indirectly, by affecting placental function in the mother; or by affecting any one of the numerous biochemical steps involved in the process of fetal development.

Animal research has found that alco hol readily crosses the placenta and can directly alter fetal development by attack ing the developing cells, thereby decreas ing cell size and cell number in several organ systems (NIAAA 1990(NIAAA , 1993)).These studies do not, however, rule out other mechanisms.Indeed research has shown that alcoholinduced damage probably results from both direct and indirect effects of the drug on the devel oping fetus.

Animal research in pregnant rats has shown rather consistently that alcohol can decrease the transport of essential nutri tional elements called amino acids across the placenta to the fetus (NIAAA 1990(NIAAA , 1993;;Schenker et al. 1990); similarly, glucose transport has been shown to be reduced.A reductio in transport of either of these building blocks could explain decreased fetal weight and other types of abnormal development.

It has been suggested that alcohol induced structural defects may be a result of an alteration in a class of compounds called prostaglandins that are important for normal fetal growth and development as well as control of umbilical blood flow.2An imbalance of prostaglandin levels, in turn, may adversely a fect de velopment in two ways: by altering cellu lar differentiation of the developing fetus and by reducing blood flow to and from the placenta.Decreased blood flow would be expected to result in a lowered supply of amino acids and oxygen to the fetus.Both decreased oxygen and decreased nutrient supply would have a negative effect on growth and development.

Technological advances now available to the researcher will permit direct quan tification of placental and umbilical blood flow and oxygen consumption.If, for example, blood flow is found to be re duced in alcoholconsuming pregnant animals, scientists can begin to address the biochemical reason for the decrea ed flow, such as an imbalance of certain prostaglandins.

Scientists also are using animal mod els to examine the effects of alcohol on chemical growth factors, which play a critical role in the regulation of cell growth and survival, and on other biologi cal mechanisms that may be involved in programmed death of brain cells (West et al. 1990).Fetal development is a omplex process that itself is not yet fully under stood.Cell signaling systems that appear to control various aspects of fetal devel opment, such as certain neurotransmitters (e.g., NmethylDaspartate [NMDA] and serotonin), retinoic acid, and cyclic AMP, may have a significant role in FAS, as may the expression of developmental genes.The cell signaling systems and the expression of developmental genes both appear to be affected by alcohol (Pullarkat and Azar 1991;Hogan 1991).


CONCLUSION

Many more potential mechanisms of action remain.This area of investigation into the basis for the teratogenic actions of alcohol is still in its infancy stage.The future looks bright for animal research to make major advances not only in the identification of potential mechanism(s) but also in the ev

uation of v
rious types of interventions.As for other medical conditions, the development of appropri ate treatment strategies for FAS can only be realized and successfully implemented when the mechanisms underlying the myriad defects related to fetal alcohol exposure are uncovered.■

Figure 1
1
Figure 1 Similarities of facial defects found in (A) humans and (B) mice exposed prenatally to alcohol.Panel C shows a control mouse fetus not exposed to alcohol.Photograph courtesy of Kathy K. Sulik.


Table 1
1
Behavioral Effects Following Prenatal Alcohol Exposure in Humans and Animals
HumansAnimalsHyperactivityIncreased activity and explorationAttention deficits, distractibilityAttention deficitsLack of inhibitionLack of inhibitionMental retardation, learning difficultiesImpaired learningImpaired ability to adjust to new stimuli or situations (impa